Recombinant DNA constructs employing site-specific recombination

ABSTRACT

Recombinant DNA constructs, for use in plants and plant cells, have site-specific recombination sites that allow assessing phenotypes and modes of action by over expression or suppression of endogenous genes. In an aspect, a single DNA construct can be switched between over expression and suppression by the action of a recombinase such as the Cre recombinase on constructs having lox recombination sites. Other useful recombination systems include the Flp/frt system, the R/Rs system, the Dre/rox system, and the GIN/gix system.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Non-Provisional patentapplication Ser. No. 12/869,806, filed on Aug. 27, 2010, which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Disclosed herein are recombinant DNA constructs utilizing site-specificrecombination technology and methods of making and using suchconstructs.

BACKGROUND OF THE INVENTION

The study of gene mode of action and effect of phenotype in plants oftenemploys transgenic plants that over express an transgenic gene andtransgenic plants that suppress a endogenous gene where the plants aretransformed using vectors to insert recombinant DNA constructs forregulating endogenous plant genes are designed to either over expressthe gene or to suppress the gene. Likewise, transgenic plants with therecombinant DNA construct to either over express or suppress the gene inplants are made with separate constructs that may not providecomparative traits due to interactions from transgene insertions atdifferent chromosomal loci.

SUMMARY OF THE INVENTION

Mode of action studies are better made using a single construct insertedin one chromosomal locus that can be switched between expression andsuppression of an endogenous gene. The invention provides recombinantDNA constructs for assessing both suppression and expression of anendogenous gene. More specifically, the recombinant DNA constructscomprise a promoter operably linked to sense-oriented DNA encoding anendogenous protein. The dual function of the recombinant DNA constructsis made possible by the presence of at least one fragment of the DNAencoding the endogenous protein in the anti-sense orientation and a pairof recombination sites which are recognized by a site-specificrecombinase. At least one of the recombination sites is located betweenthe DNA encoding an endogenous protein and the antisense fragment. Thelocation of the other recombination site of the pair can vary dependingof the design as illustrated below. The recombinant DNA constructs alsocomprises DNA providing a polyadenylation sequence.

As illustrated herein depending of the design, stable integration of arecombinant DNA construct into plant cells results in either expressionor suppression of the DNA encoding an endogenous protein. Introductionof a recombinase that recognizes the recombination sites switches to thealternative function of the construct. A plant comprising a recombinantDNA construct of this invention will, depending on design, have anoriginal function that either expresses an endogenous gene or suppressesthe endogenous gene. When a plant is transformed with a vectorcontaining this construct and then crossed with a plant containing arecombinase that recognizes the recombination sites,recombinase-mediated excision will remove the DNA between therecombination sites allowing for a change in original function. Forinstance, when the original function is suppression of the endogenousgene the presence of recombinase will switch the function to allowexpression of DNA encoding the endogenous protein.

In one aspect the recombinant DNA construct comprises a spacer that islocated between the pair of recombination sites. The spacer has a lengthsufficient to prevent fold back and self-hybridization in RNAtranscribed from the recombinant DNA construct to maintain expression ofthe DNA encoding the endogenous protein. Any of a number of siterecognizing recombinases can be used in the practice of this inventionincluding Cre recombinase with lox sites, Flp recombinase with frtsites, R recombinase with R sites, Dre recombinase with rox sites andGin recombinase with gix sites. When a transformed plant containing thisconstruct is crossed to a plant expressing Cre recombinase, Cre-mediatedrecombination at the first pair recombination sites will remove thespacer and will switch the function to allow suppression of DNA encodingthe endogenous protein.

In aspects of the invention the recombinant DNA constructs furthercomprise DNA for one or more other elements, such as DNA encoding aselectable marker, DNA encoding a recombinase and/or DNA for a secondpair of site-specific recombination sites. The DNA for encodingselectable marker, DNA encoding the recombinase or both can be locatedeither between the first pair of site-specific recombination sites orsecond pair of site-specific recombination sites or both. One importantaspect of the invention that a promoter operably linked to DNA encodingthe recombinase is not constitutive. Useful promoters are chemicallyinducible promoters that are regulated by external agent. Stableintegration of a recombinant DNA construct into plant cells results insuppression the DNA encoding protein and allow evaluation ofsuppression. When a transformed plant contains DNA for a selectablemarker between recognition sites, e.g. between a second pair ofrecognition sites, is crossed to a plant expressing a recombinase eitherof the pairs of the recombination sites can be involved inrecombination, recombination at the second pair of recombination siteswill remove the DNA for the selectable marker allowing marker freesuppression of the protein When a transformed plant contains antisenseDNA encoding the endogenous protein and DNA for a selectable markerbetween recognition sites, e.g. between a first pair of recognitionsites, is crossed to a plant expressing a recombinase, recombination atthe first pair of recombination sites will remove antisense DNA encodingthe endogenous protein and DNA for the selectable marker allowing markerfree expression.

When a transformed plant contains DNA for a selectable marker and DNAfor a recombinase between recognition sites, e.g. between a second pairof recognition sites, expressing the recombinase either of the pairs ofthe recombination sites can be involved in recombination, recombinationat the second pair of recombination sites will remove the DNA for theselectable marker and DNA for the recombinase allowing marker freesuppression of the protein. When a transformed plant contains antisenseDNA for a endogenous protein, DNA for the selectable marker and DNA forthe recombinase between recognition sites, e.g. between a first pair ofrecognition sites, expressing the recombinase, recombination at thefirst pair of recombination sites will remove antisense DNA forendogenous protein, DNA for the selectable marker and DNA for therecombinase allowing marker free expression. In one aspect transformedplant contains DNA for a recombinase operably linked to a constitutivepromoter between recognition sites, e.g. between a second pair ofrecognition sites, expressing the recombinase either of the pairs of therecombination sites can be involved in recombination, recombination atthe second pair of recombination sites will remove the DNA for therecombinase allowing suppression of the protein with selectable marker.When transformed plant contains antisense DNA for a endogenous protein,DNA for the recombinase between recognition sites, e.g. between a firstpair of recognition sites, expressing a recombinase, recombination atthe first pair of recombination sites will remove antisense DNA forendogenous protein and DNA for the recombinase allowing expression ofthe protein with selectable marker.

In another aspect the recombinant DNA constructs further comprise aninverted repeat of a DNA that serves as a screenable marker which DNA islocated adjacent to the antisense fragment. In useful aspects of theinvention the inverted repeat DNA functions to suppress a gene thatprovides a visible phenotype, e.g. DNA for suppressing the opaque 2gene. Stable integration of recombinant DNA into plant/cells will allowa visual phenotype indicating desired suppression of DNA encoding for adetectable marker. In some aspects of the invention the DNA for encodingselectable marker and DNA encoding site specific recombinase are locatedbetween the second pair of site-specific recombination sites and theantisense fragment, the inverted repeat of DNA encoding for a screenablemarker, DNA for encoding selectable marker and DNA encoding a sitespecific recombinase are located between the first pair of site-specificrecombination sites. In other aspects of the invention the DNA encodingsite specific recombinase is located between the second pair ofsite-specific recombination sites and the antisense fragment, theinverted repeat of DNA encoding for a screenable marker, and DNAencoding a site specific recombinase are located between the first pairof site-specific recombination sites.

This invention provides a method for generating a transformed plant celland plant. This invention also provides a method for generating progenyplant seed by crossing a first plant containing the recombinant DNAconstruct with a second plant containing DNA expressing the first sitespecific recombinase to produce a population of seed that will produceplants that will express DNA encoding protein and plants that willsuppress the production of said protein with or without a selectablemarker.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-4 illustrate the design of recombinant DNA constructs and theiruse as described in the Examples. FIGS. 1a, 1b, and 1c are described indetail in Example 1. FIG. 1a depicts a recombinant DNA constructcontaining a promoter (P) operably linked to a DNA having a nucleotidesequence of interest (SOI) encoding a protein of interest for expressionor suppression, a fragment in antisense orientation of the SOI having atleast 21 nt, a pair of loxP recombination sites surrounding andbracketing the antisense fragment, and a DNA element providingpolyadenylation sequence (T). FIG. 1b depicts an alternative arrangementof the construct shown in FIG. 1a where the promoter (P) is followed bythe antisense fragment and then the SOI, wherein a pair of recombination(loxP) sites bracket the antisense fragment. FIG. 1c depicts arecombinant DNA construct containing a promoter (P) operably linked tothe SOI, a loxP recombination site, a DNA element providing a firstpolyadenylation sequence (T1), a spacer with a length sufficient toprevent fold back and self-hybridization in RNA transcribed from therecombinant DNA construct, a second loxP recombination site, anantisense fragment of the SOI having at least 21 nt, and a DNA elementproviding a second polyadenylation sequence (T2), wherein the pair ofloxP recombination sites brackets the polyadenylation sequence T1 andthe spacer. FIGS. 2a and 2b are described in detail in Example 2. FIG.2a depicts a recombinant DNA construct with two expression cassettes,wherein the construct contains a promoter (P1) operably linked to theSOI, a first loxP recombination site, an antisense fragment of the SOI,DNA providing a first polyadenylation sequence (T1), a first lox511recombination site, a second promoter (P2) operably linked to a DNAencoding a selectable marker, a second loxP recognition site, DNAproviding a second polyadenylation sequence (T2), and a second lox511recognition site, wherein the first pair of recombination (loxP) sitesbrackets the antisense fragment, the first polyadenylation sequence(T1), the first lox511 site, the second promoter (P2) and the DNAencoding selectable marker, and wherein the second pair of recombination(lox511) sites brackets the selectable marker expression cassette andthe second loxP site. FIG. 2b depicts a recombinant DNA constructsimilar to that of FIG. 2a but having an additional DNA encoding ascreenable marker (e. g., an inverted repeat of DNA for suppressing acorn opaque 2 gene). FIGS. 3a and 3b are described in detail in Example3. FIG. 3a depicts a recombinant DNA construct containing 3 cassettes: apromoter (P1) operably linked to the SOI, a loxP recombination site,antisense fragment of the SOI, DNA providing a first polyadenylationsequence (T1), a lox511 recombination site, a second promoter (P2)operably linked to a DNA encoding a selectable marker, DNA providing asecond polyadenylation sequence (T2), a third promoter (P3) that isnon-constitutive and is operably linked to DNA encoding a Crerecombinase, second loxP recombination site, DNA providing a thirdpolyadenylation sequence (T3), and a second lox511 recognition site. Thefirst pair of recombination sites (the loxP sites) brackets theantisense fragment, the first polyadenylation sequence (T1), the firstlox511 site, the selectable marker expression cassette, the thirdpromoter (P3) and the DNA encoding for Cre recombinase. The second pairof recombination sites (the lox511 sites) brackets the selectable markerexpression cassette, the Cre recombinase expression cassette, and thesecond loxP site. FIG. 3b depicts a recombinant DNA construct similar tothat of FIG. 3a but having an additional DNA encoding a screenablemarker (e. g., an inverted repeat of DNA for suppressing a corn opaque 2gene located adjacent to the antisense fragment). FIGS. 4a and 4b aredescribed in detail in Example 4. FIG. 4a depicts a recombinant DNAconstruct containing three cassettes: a promoter (P1) operably linked tothe SOI, a first loxP recombination site, an antisense fragment of theSOI, DNA providing a first polyadenylation sequence (T1), a first lox511recombination site, a constitutive promoter (P3) operably linked to DNAencoding a Cre recombinase, a second loxP recombination site, DNAproviding a third polyadenylation sequence (T3), a second lox511recognition site, a promoter (P2) operably linked to a DNA encoding aselectable marker, and DNA providing a second polyadenylation sequence(T2). The first pair of recombination sites (the loxP sites) bracketsthe antisense fragment, the first polyadenylation sequence (T1), thefirst lox511 site, the constitutive promoter (P3), and the DNA encodingfor Cre recombinase. The second pair of recombination sites (the lox511sites) brackets the Cre recombinase expression cassette and the secondloxP site. FIG. 4b depicts a recombinant DNA construct similar to thatof FIG. 4a but having an additional DNA encoding a screenable marker (e.g., an inverted repeat of DNA for suppressing a corn opaque 2 genelocated adjacent to the antisense fragment).

DETAILED DESCRIPTION

As used herein, the term “recombinant DNA construct” means a man-made,non-natural DNA molecule.

As used herein, the term “promoter” means a DNA molecule that isinvolved in recognition and binding of RNA polymerase, commonly an RNApolymerase II, and other proteins (such as trans-acting transcriptionfactors) to initiate transcription. Promoters may be defined asconstitutive, i.e. generally always active, and or as inducible i.e.conditionally active i.e. active in presence of certain exogeneouscomponent A promoter may be initially isolated from the 5′ untranslatedregion (5′ UTR) of a genomic copy of a gene. Alternately, promoters maybe synthetically produced or manipulated DNA molecules. Promoterfragments may exhibit promoter activity and may be useful in therecombinant DNA constructs of the invention.

As used herein, the term “3′ transcription termination molecule” refersto a DNA molecule that is used during transcription to produce the 3′untranslated region (3′ UTR) of an messenger RNA (mRNA) molecule. The 3′untranslated region of an mRNA molecule may be generated by specificcleavage and 3′ polyadenylation producing what is known as a polyA tail.A 3′ UTR may be operably linked to and located downstream of a DNAencoding protein and may include polynucleotides that provide apolyadenylation signal and other regulatory signals capable of affectingtranscription, mRNA processing, or gene expression. PolyA tails arethought to function in mRNA stability and in initiation of translation.

As used herein, the term “marker” means any DNA molecule that allows theidentification of the presence of recombinant DNA construct. The markercan be selectable or screenable. A “selectable marker” refers to amarker that expresses a protein that allows survival of cell expressingthe marker. A “screenable marker” refers to a marker that expresses aprotein that provides a visible phenotype for identifying the presenceof the marker. Useful selectable markers include those expressingprotein that confers resistance to antibiotics such as kanamycin(nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) orresistance to herbicides such as glufosinate (bar or pat) and glyphosate(EPSPS). Useful screenable markers include those expressing protein thatconfers visual phenotype such as a gene expressing a colored orfluorescent protein such as a luciferase or green fluorescent protein(GFP) or a gene expressing a beta-glucuronidase or uidA gene (GUS) forwhich various chromogenic substrates are known, and those suppressingthe native protein such as maize opaque 2 protein.

A recombinant DNA construct can include one or more expressioncassettes, i.e. recombinant DNA for expressing specific mRNA andtypically comprising a promoter element, a DNA sequence of interest, and3′ UTR. Further, an expression cassette can be arranged in anyorientation capable of either facilitating expression or suppression ofa gene of interest

As used herein, “site specific recombination sites” means a pair of DNAsegments that is recognized by a site specific recombinase in a processthat allows the excision of the DNA between the pair of DNA segments.For instance, Cre recombinase recognizes either loxP recombination sitesor lox511 recombination sites which are hetero-specific, which meansthat loxP and lox511 do not recombine together. The Cre/lox system isdisclosed by Odell et al., Plant Physiol, 106(2): 447-58 (1994) FLPrecombinase recognizes frt recombination sites as disclosed by Lyznik etal., Nucleic Acids Res 24(19), 3784-9 (1996). the R recombinaserecognizes Rs recombination sites as disclosed by Onounchi et al.,Nucleic Acid Res., 19:6373-6378 (1991). The Dre recombinase recognizesrox sites as disclosed in U.S. Pat. No. 7,422,889. And, Gin recombinaserecognizes gix sites as disclosed by Maeser et al., Mol. Gen. Genet.230: 170-176 (1991).

An aspect of the invention is described by reference to a first pair ofsite specific recombination sites, e.g. loxP, recognized by asite-specific recombinase, e.g. Cre recombinase, and a second pair sitespecific recombination sites, e.g. lox511, also recognized by thesite-specific Cre recombinase. An expression cassette can be arranged inany order or orientation that results in either the expression orsuppression of the target gene of interest following recombination atone set of the heterospecific lox sites. In other aspects there is acombination of recombination systems.

As used herein, the term “operably linked” is used to describe afunctional relationship between a promoter and transcribable DNA andbetween a transcribable DNA and 3′ polyadenylation sequence.

A sequence of interest (SOI) represents polynucleotides from a gene ofinterest (GOI) where the polynucleotides are DNA encoding a protein. Thesequence of interest is a component of the recombinant DNA constructsthat is expressed in embodiments of the invention to investigate whetheran expressed protein coded by an endogenous gene of interest is capableof contributing to producing a desirable phenotype or trait. In onespecific aspect the invention is illustrated by using a sequence ofinterest that encodes the maize opaque 2 (O2) protein. The sequence ofinterest can be a nucleic acid sequence that causes the targetedexpression, typically over-expression, of an endogenous gene. A sequenceof interest is also used to design the gene suppression components ofthe recombinant DNA constructs that produce double-stranded RNA that cancause the inhibition of expression of an endogenous gene via naturalgene silencing mechanisms. For instance, the DNA encoding proteinderived from the sequence of interest (SOI) is advantageously arrangedin an antisense orientation proximate to the sense oriented DNA butseparated from the sense oriented DNA by recombination sites to becapable of either facilitating expression or suppression of anendogenous gene of interest depending on the presence of a recombinase.

Plant Cell Transformation Methods

Numerous methods for transforming chromosomes in a plant cell nucleuswith recombinant DNA constructs are known in the art and are used inmethods of preparing a transgenic plant cell and plant as generallydescribed herein. Two effective methods for such transformation areAgrobacterium-mediated transformation and microprojectile bombardment.Microprojectile bombardment methods are illustrated in U.S. Pat. No.5,015,580 (soybean); U.S. Pat. No. 5,550,318 (corn); U.S. Pat. No.5,538,880 (corn); U.S. Pat. No. 5,914,451 (soybean); U.S. Pat. No.6,399,861 (corn); U.S. Pat. No. 6,153,812 (wheat) and U.S. Pat. No.6,365,807 (rice) and Agrobacterium-mediated transformation is describedin U.S. Pat. No. 5,159,135 (cotton); U.S. Pat. No. 5,824,877 (soybean);U.S. Pat. No. 5,463,174 (canola); U.S. Pat. No. 5,591,616 (corn); U.S.Pat. No. 5,846,797 (cotton); U.S. Pat. No. 6,384,301 (soybean), U.S.Pat. No. 7,026,528 (wheat) and U.S. Pat. No. 6,329,571 (rice), and USPatent Application Publication 2001/0042257 A1 (sugar beet), all ofwhich are incorporated herein by reference for enabling the productionof transgenic plants. Transformation of plant material is practiced intissue culture on a nutrient media, i.e. a mixture of nutrients thatwill allow cells to grow in vitro. Recipient cell targets include, butare not limited to, meristem cells, hypocotyls, calli, immature embryosand gametic cells such as microspores, pollen, sperm and egg cells.Callus may be initiated from tissue sources including, but not limitedto, immature embryos, hypocotyls, seedling apical meristems, microsporesand the like. Cells containing a transgenic nucleus are grown intotransgenic plants.

In addition to direct transformation of a plant material with arecombinant DNA, a transgenic plant cell can be prepared by crossing afirst plant having cells with recombinant DNA construct with a secondplant lacking the recombinant construct. For example, recombinant DNAcan be introduced into a nucleus from a first plant line that isamenable to transformation to transgenic nucleus in cells that are growninto a transgenic plant which can be crossed with a second plant linefor the introgression of the recombinant DNA into the second plant line.A transgenic plant with recombinant DNA constructs of this invention canbe crossed with plant line containing DNA for expressing a recombinasethat recognizes the recombination sites in the transgenic plant tomodify the recombinant DNA construct by recombination. The progeny ofthis cross will segregate such that some of the plants will carry theDNA from both parental nucleus and some will carry DNA from one parentalnucleus; such plants can be identified by markers associated withparental recombinant DNA, e.g. marker identification by analysis forrecombinant DNA or, in the case where a selectable marker is linked tothe recombinant. If desired, progeny plants carrying DNA from bothparents can be crossed back into one parent line multiple times, forexample usually 6 to 8 generations, to produce a progeny plant withsubstantially the same genotype as one original transgenic parental linebut for the recombinant DNA of the other transgenic parental line.

In the practice of transformation DNA is typically introduced into onlya small percentage of target plant cells in any one transformationexperiment. Marker genes are used to provide an efficient system foridentification of those cells that are stably transformed by receivingand integrating a recombinant DNA molecule into their genomes. Preferredmarker genes provide selective markers which confer resistance to aselective agent, such as an antibiotic or a herbicide. Any of theherbicides to which plants of this invention may be resistant are usefulagents for selective markers. Potentially transformed cells are exposedto the selective agent. In the population of surviving cells will bethose cells where, generally, the resistance-conferring gene isintegrated and expressed at sufficient levels to permit cell survival.Cells may be tested further to confirm stable integration of theexogenous DNA. Markers which provide an ability to visually screentransformants can also be employed.

Plant cells that survive exposure to the selective agent, or plant cellsthat have been scored positive in a screening assay, may be cultured inregeneration media and allowed to mature into plants. Developingplantlets regenerated from transformed plant cells can be transferred toplant growth mix, and hardened off, for example, in an environmentallycontrolled chamber prior to transfer to a greenhouse or growth chamberfor maturation. Plants are regenerated from about 6 weeks to 10 monthsafter a transformant is identified, depending on the initial tissue, andplant species. Plants may be pollinated using conventional plantbreeding methods known to those of skill in the art and seed produced,for example self-pollination is commonly used with transgenic corn. Theregenerated transformed plant or its progeny seed or plants can betested for expression of the recombinant DNA.

Transgenic Plants and Seeds

Transgenic plants derived from transgenic plant cells having atransgenic nucleus of this invention are grown to generate transgenicplants having an enhanced trait as compared to a control plant andproduce transgenic seed Such plants with enhanced traits are identifiedby selection of transformed plants or progeny seed for the enhancedtrait. For efficiency a selection method is designed to evaluatemultiple transgenic plants (events) comprising the recombinant DNA, forexample multiple plants from 2 to 20 or more transgenic events.Transgenic plants grown from transgenic seed provided herein that willproduce plants that will express DNA encoding protein and plants thatwill suppress the production of said protein with or without aselectable marker.

EXAMPLE 1

This example illustrates the design of a recombinant DNA construct thatis useful in the practice of this invention. With reference to FIG. 1a ,there is shown a recombinant DNA construct containing a promoter (P)operably linked to a DNA having a nucleotide sequence encoding a proteinof interest for expression or suppression, i.e. designated a sequence ofinterest (SOI), a fragment in antisense orientation of the SOI having atleast 21 nt, a pair of loxP recombination sites surrounding andbracketing the antisense fragment, and a DNA element providingpolyadenylation sequence (T). The pair of loxP recombination sites isrecognized by a Cre site-specific recombinase. The recombinant DNAconstruct is stably transformed into a corn cell which is regenerated toa transformed corn plant. In the transformed corn plant containing thisconstruct the production of a double stranded RNA (dsRNA) transcriptprovides suppression of the endogenous DNA encoding protein. Without Crerecombinase, the construct is transcribed to produce a hairpin RNA thatsuppressed the production of the endogenous protein. When Crerecombinase is present, e.g. from crossing with a corn plant expressingthe recombinase, the DNA bracketed by the loxP recombination sites isexcised, i.e. the antisense fragment is removed, allowing the productionof a transcript encoding the protein-. This allows the evaluation of acorn plant with over expression of the endogenous protein. The DNAconstruct is also transformed into cotton, rice, wheat and soybean cellswhich are regenerated inot plants to provide evaluation of expressionand suppression of an endogenous gene from a construct in the samegenetic locus.

With reference to FIG. 1b , there is shown an alternative arrangement ofthe construct shown in FIG. 1a to achieve a similar outcome intransgenic plants and cells where the promoter (P) is followed by theantisense fragment and then the DNA encoding protein (SOI); and a pairof loxP recombination sites surround and bracket the antisense fragmentas in FIG. 1 a.

With reference to FIG. 1c there is shown a recombinant DNA constructcontains a promoter (P) operably linked to a DNA having a nucleotidesequence encoding a protein of interest for expression or suppression,i.e. designated a sequence of interest (SOI), a loxP recombination site,a DNA element providing polyadenylation sequence (T1), a spacer with alength sufficient to prevent fold back and self-hybridization in RNAtranscribed from the recombinant DNA construct, second loxPrecombination site, antisense fragment of the SOI having at least 21 nt,and a DNA element providing a second polyadenylation sequence (T2). Thepair of loxP recombination sites is surrounding and bracketing thepolyadenylation sequence T1 and the spacer. Transformed plants and plantcells containing this construct result in enhanced expression ofsequence of interest (SOI). Without Cre recombinase, the construct istranscribed to over express the endogenous protein. When Cre recombinaseis present, e.g. from crossing with a plant expressing the recombinase,the DNA bracketed by the loxP recombination sites is excised, i.e. thefirst polyadenylation sequence, T1 and the spacer fragment are removed,allowing the evaluation of a plant with suppression of the endogenousprotein.

EXAMPLE 2

With reference to FIG. 2a there is shown a recombinant DNA constructwith two expression cassettes. More specifically the construct containsa promoter (P1) operably linked to DNA having a nucleotide sequenceencoding a protein of interest for expression or suppression, i.e.designated a sequence of interest (SOI), a loxP recombination site, anantisense fragment of the DNA encoding the endogenous protein, DNAproviding a first polyadenylation sequence (T1), a lox511 recombinationsite, a second promoter (P2) operably linked to a DNA encoding aselectable marker, a second loxP recognition site, DNA providing secondpolyadenylation sequence (T2) and a second lox511 recognition site. Thefirst pair of recombination sites, i.e. the loxP sites, is surroundingand bracketing the antisense fragment, the first polyadenylationsequence (T1), the first lox511 site, the second promoter (P2) and theDNA encoding selectable marker. The second pair of recombination sitesi.e. the lox511 sites, is surrounding and bracketing the selectablemarker expression cassette, and the second loxP site. The recombinantDNA construct is stably transformed into a corn cell which isregenerated to a transformed corn plant. In the transformed corn plantscontaining this construct results in production of a double stranded RNA(dsRNA) transcript that provides suppression of the endogenous DNAencoding protein (SOI) with expression of selectable marker protein.When transformed plant is crossed to a plant expressing a Crerecombinase, either of the pairs of the recombination sites can beinvolved in recombination. When the loxP sites are recombined, antisensefragment, first polyadenylation sequence (T1), the first lox511 site,the second promoter (P2) and the DNA encoding selectable marker areremoved allowing for over expression of the endogenous protein. When thelox511 sites are recombined, the selectable marker expression cassetteis removed allowing for transcription of RNA that forms a dsRNA forsuppression of the endogenous protein.

The DNA construct is also transformed into cotton, rice, wheat andsoybean cells which are regenerated into plants to provide evaluation ofexpression and suppression of an endogenous gene from a construct in thesame genetic locus.

With reference to FIG. 2b there is shown a recombinant DNA constructsimilar to that of FIG. 2a with an additional DNA encoding a screenablemarker, e.g., as an inverted repeat of DNA for suppressing corn opaque 2gene. The inverted repeat DNA is located adjacent to the antisensefragment. The inverted repeat DNA functions to suppress a gene thatprovides a visible phenotype, e.g. DNA for suppressing the opaque 2gene. Stable integration of recombinant DNA into plant will allow avisual phenotype indicating desired suppression of DNA encoding for ascreenable marker. When transformed plant is crossed to a plantexpressing a Cre recombinase, either of the pairs of the recombinationsites can be involved in recombination. When the loxP sites arerecombined, antisense fragment, inverted repeat of DNA, firstpolyadenylation sequence (T1), the first lox511 site, the secondpromoter (P2) and the DNA encoding selectable marker are removedallowing for over expression the endogenous protein. When the lox511sites are recombined, the selectable marker expression cassette isremoved allowing suppression of the gene that provides visiblephenotype, e.g. DNA for suppressing the opaque 2 gene.

EXAMPLE 3

With reference to FIG. 3a there is shown a recombinant DNA constructcontaining three cassettes. More specifically, the construct contains aa promoter (P1) operably linked to DNA encoding protein i.e. designateda sequence of interest (SOI), a loxP recombination site, antisensefragment of the DNA encoding protein, DNA providing a firstpolyadenylation sequence (T1), a lox511 recombination site, a secondpromoter (P2) operably linked to a DNA encoding a selectable marker, DNAproviding second polyadenylation sequence (T2), a third promoter (P3)operably linked to a DNA encoding a Cre recombinase, second loxPrecombination site, DNA providing third polyadenylation sequence (T3)and a second lox511 recognition site. It is important that the thirdpromoter (P3) not be constitutive. Useful P3 promoters are chemicallyinducible promoter that are regulated by external agent. See Wang etal., “Chemically regulated expression nsystems and their applications ntransgenic plants”, Transgenic Research, 12:529-540, 2003 for adisclosure of copper-inducible promoters and glucocorticoid-induciblepromoters. The first pair of recombination sites, i.e. the loxP sites,is surrounding and bracketing the antisense fragment, the firstpolyadenylation sequence (T1), the first lox511 site, selectable markerexpression cassette, third promoter (P3) and DNA encoding for Crerecombinase. The second pair of recombination sites, i.e. the lox511sites, is surrounding and bracketing the selectable marker expressioncassette, the Cre recombinase expression cassette and the second loxPsite. The recombinant DNA construct is stably transformed into a corncell which is regenerated to a transformed corn plant. In thetransformed corn plants containing this construct result in productionof a double stranded RNA (dsRNA) transcript that provides suppression ofthe endogenous DNA encoding protein (SOI) with expression of selectablemarker protein. When transformed plant expressing a Cre recombinase byactivation of P3, either of the pairs of the recombination sites can beinvolved in recombination. When the loxP sites are recombined, antisensefragment, first polyadenylation sequence (T1), the first lox511 site,selectable marker expression cassette, third promoter (P3) and the DNAencoding for Cre recombinase are removed allowing for over expression ofthe endogenous protein. When the lox511 sites are recombined, theselectable marker expression cassette and Cre recombinase expressioncassette are removed allowing for transcription of RNA that forms adsRNA for suppression of the endogenous protein.

The DNA construct is also transformed into cotton, rice, wheat andsoybean cells which are regenerated into plants to provide evaluation ofexpression and suppression of an endogenous gene from a construct in thesame genetic locus.

With reference to FIG. 3b there is shown a recombinant DNA constructsimilar to that of FIG. 3a with an additional DNA encoding a screenablemarker, e.g., as an inverted repeat of DNA for suppressing corn opaque 2gene. The inverted repeat DNA is located adjacent to the antisensefragment. The inverted repeat DNA functions to suppress a gene thatprovides a visible phenotype, e.g. DNA for suppressing the opaque 2gene. Stable integration of recombinant DNA into plant will allow avisual phenotype indicating desired suppression of DNA encoding for ascreenable marker. When transformed plant expressing a Cre recombinaseby activation of P3, either of the pairs of the recombination sites canbe involved in recombination, either of the pairs of the recombinationsites can be involved in recombination. When the loxP sites arerecombined, antisense fragment, inverted repeat of DNA, firstpolyadenylation sequence (T1), the first lox511 site, selectable markerexpression cassette, third promoter (P3) and the DNA encoding Crerecombinase are removed allowing for over expression of the gene thatprovides visible phenotype, e.g. DNA for the opaque 2 gene. When thelox511 sites are recombined, the selectable marker expression cassette,third promoter (P3) and the DNA encoding for Cre recombinase are removedallowing suppression of the gene that provides visible phenotype, e.g.DNA for suppressing the opaque 2 gene. This allows the evaluation ofenhanced expression and suppression for the DNA encoding screenablemarker.

EXAMPLE 4

With reference to FIG. 4a there is shown a recombinant DNA constructcontaining three cassettes. More specifically, the construct contains apromoter (P1) operably linked to DNA encoding protein, i.e. designated asequence of interest (SOI), a loxP recombination site, antisensefragment of the DNA encoding protein, DNA providing a firstpolyadenylation sequence (T1), a lox511 recombination site, a promoter(P3) operably linked to a DNA encoding a Cre recombinase, second loxPrecombination site, DNA providing polyadenylation sequence (T3), secondlox511 recognition site, a promoter (P2) operably linked to a DNAencoding a selectable marker, and a polyadenylation sequence (T2). Anadvantage of the construct illustrated in FIG. 4 is that the thirdpromoter (P3) is constitutive. The first pair of recombination sites,i.e. the loxP sites, is surrounding and bracketing the antisensefragment, the first polyadenylation sequence (T1), the first lox511site, promoter (P3) and DNA encoding for Cre recombinase. The secondpair of recombination sites, i.e. the lox511 sites, is surrounding andbracketing the Cre recombinase expression cassette along with secondloxP site. The recombinant DNA construct is stably transformed into acorn cell which is regenerated to a transformed corn plant. In thetransformed corn plants containing this construct result in productionof a double stranded RNA (dsRNA) transcript that provides suppression ofthe endogenous DNA encoding protein (SOI) with expression of selectablemarker protein. When a transformed plant expressing a Cre recombinase,either of the pairs of the recombination sites can be involved inrecombination. When the loxP sites are recombined, antisense fragment,first polyadenylation sequence (T1), the first lox511 site, promoter(P3), and DNA encoding for Cre recombinase are removed allowing for overexpression of the endogenous protein. When the lox511 sites arerecombined, the Cre recombinase expression cassette is removed allowingfor transcription of RNA that forms a dsRNA for suppression of theendogenous protein.

The DNA construct is also transformed into cotton, rice, wheat andsoybean cells which are regenerated into plants to provide evaluation ofexpression and suppression of an endogenous gene from a construct in thesame genetic locus.

With reference to FIG. 4b there is shown a recombinant DNA constructsimilar to that of FIG. 4a with an additional DNA encoding a screenablemarker comprising an inverted repeat of DNA for suppressing corn opaque2 gene to is the inverted repeat DNA is located adjacent to theantisense fragment. Stable integration of the recombinant DNA into aplant will allow a visual phenotype with the screenable markerindicating the suppression of the endogenous gene. When a transformedplant expressing a Cre recombinase, either of the pairs of therecombination sites can be involved in recombination. When the loxPsites are recombined, antisense DNA encoding the endogenousprotein(SOI), inverted repeat, and DNA encoding recombinase are removedallowing expression of DNA encoding protein (SOI) without indicationfrom the screenable marker When lox511 sites are recombined, Crerecombinase expression cassette and second loxP site are removedallowing suppression of the protein (SOI) with visible indication fromthe selectable marker.

We claim:
 1. A recombinant DNA molecule comprising: (a) a first DNAsegment comprising (i) a first promoter operably linked to asense-oriented sequence of interest; (ii) a first member of a first pairof recombination sites; (iii) an antisense fragment of said sequence ofinterest ; (iv) DNA providing a first polyadenylation sequence; and (v)a first member of a second pair of recombination sites; (b) a second DNAsegment comprising (i) a second promoter operably linked to DNA encodinga selectable marker; and (ii) DNA providing a second polyadenylationsequence; (c) a second member of said first pair of recombination sites,located 3′ to said DNA encoding a selectable marker; and (d) a secondmember of said second pair of recombination sites, located 3′ to saidDNA providing a second polyadenylation sequence, wherein said first andsecond pairs of recombination sites are different.
 2. The recombinantDNA molecule of claim 1, wherein said second member of said first pairof recombination sites is located between said DNA encoding a selectablemarker and said DNA providing a second polyadenylation sequence, andwherein said second member of said second pair of recombination sites islocated 3′ and adjacent to said DNA providing a second polyadenylationsequence.
 3. The recombinant DNA molecule of claim 1, further comprisinga third segment comprising (i) a third promoter operably linked to DNAencoding a site-specific recombinase; and (ii) DNA providing a thirdpolyadenylation sequence, wherein said second member of said first pairof recombination sites is located between said DNA encoding saidsite-specific recombinase and said DNA providing a third polyadenylationsequence, and wherein said second member of said second pair ofrecombination sites is located 3′ and adjacent to said DNA providing athird polyadenylation sequence.
 4. The recombinant DNA molecule of claim1, wherein said recombinant DNA molecule is capable of suppressing theexpression of an endogenous gene comprising said sequence of interest ina cell in the absence of a site-specific recombinase that recognize saidfirst or second pair of recombination sites.
 5. The recombinant DNAmolecule of claim 1, wherein the recombination of said first pair ofrecombination sites excises said DNA encoding a selectable marker andenhance the expression of said sequence of interest.
 6. The recombinantDNA molecule of claim 1, wherein the recombination of said second pairof recombination sites excises said DNA encoding a selectable marker andsuppress the expression of an endogenous gene comprising said sequenceof interest.
 7. The recombinant DNA molecule of claim 1, wherein saidsequence of interest encodes a protein of interest.
 8. The recombinantDNA molecule of claim 1, wherein said first and second pairs ofrecombination sites are heterospecific.
 9. The recombinant DNA moleculeof claim 1, wherein said first or second pair of recombination sites are1oxP or 1ox511recombination sites.
 10. The recombinant DNA molecule ofclaim 1, wherein said first and second pairs of recombination sites areselected from the group consisting of lox sites for recombination withCre recombinase, Frt sites for recombination with Flp recombinase, Rssites for recombination with R recombinase, rox sites for recombinationwith Dre recombinase, and gix sites for recombination with Ginrecombinase.
 11. The recombinant DNA molecule of claim 1, wherein saidantisense fragment of said sequence of interest is at least 21nucleotides in length.
 12. The recombinant DNA molecule of claim 1,further comprising DNA encoding a visible marker.
 13. The recombinantDNA molecule of claim 12, wherein said visible marker is adjacent toantisense fragment of said sequence of interest.
 14. The recombinant DNAmolecule of claim 12, wherein said visible marker is from suppression ofa opaque 2 gene.
 15. The recombinant DNA molecule of claim 3, whereinsaid third promoter is selected from the group consisting of aconstitutive promoter, a non-constitutive promoter, an induciblepromoter, and a chemically inducible promoter.
 16. A transgenic plantcomprising the recombinant DNA molecule of claim
 1. 17. The transgenicplant of claim 16, wherein said plant is selected from the groupconsisting of soybean, corn, wheat, rice, cotton, canola, and sugarbeet.
 18. The transgenic plant of claim 16, said transgenic plantcomprising an altered property relative to a plant lacking saidrecombinant DNA molecule.
 19. A method of producing a population oftransgenic plants, said method comprising: transforming a plant cellwith the recombinant DNA molecule of claim 1, producing a primarytransgenic plant from said plant cell, collecting transgenic seeds fromsaid primary transgenic plant, and growing said transgenic seeds toproduce a population of transgenic plants, wherein said population oftransgenic plants comprise both plants with overexpression of saidsequence of interest and plants with suppression of an endogenous genecomprising said sequence of interest.
 20. The method of claim 19,further comprising: crossing said primary transgenic plant with anotherplant expressing a site-specific recombinase.
 21. A population oftransgenic plants produced from a common parent plant, wherein saidpopulation comprises plants having overexpression of a sequence ofinterest and plants having suppression of an endogenous gene comprisingsaid sequence of interest, wherein said common parent plant comprisessaid recombinant DNA molecule of claim 1, wherein the parent plant ishomozygous for said recombinant DNA molecule.